The following points highlight the six full proof techniques of chromatography. The techniques are: 1. Partition Chromatography 2. Ion Exchange Chromatography 3. Adsorption Chromatography 4. Gel Permeation Chromatography 5. Gas Chromatography Principle 6. High Performance Liquid Chromatography (HPLC).
Technique # 1. Partition Chromatography:
Two immiscible liquid phases are passed through the supporting phase-containing materials. This is also called Liquid-Liquid Chromatography. Another type of Partition Chromatography is Gas-Liquid Partition Chromatography, where carrier gases such as N2, helium, argon, H2, and CO2 are not carrier gases used as mobile phase.
In Partition Chromatography one solvent acts as stationary and the other as mobile phase:
The materials of Chromatographic phase are:
(1) Silica gel,
(5) Dextrin, and
(6) Rubber and Chlrorinated rubber.
This method is used for the following analysis:
1. Amino acids and peptides separation;
2. Protein separation;
3. Separation of carbohydrates;
4. Organic acids;
Technique # 2. Ion Exchange Chromatography:
In this method ion exchange materials are used as the stationary phase in chromatography. A variety of materials have been used as ion exchanger.
Thus most important categories of ion exchangers are:
(a) Natural and artificial siliceous minerals;
(b) Sulphonated carbonaceous materials;
(c) Synthetic polymers;
(d) Derivatives of cellulose;
Technique # 3. Adsorption Chromatography:
In this method certain adsorbents are used for separation technique. Many substances of widely different chemical nature have been used as adsorbents.
The essential apparatus for a chromatographic separation is very simple. It consists of a tube of suitable dimensions, with means for supporting the packed adsorbent and openings for the admission and collection of mobile phase. Recently, multiple coupled filter column is used for this purpose.
The major types of adsorbents are:
(a) Polar: Alumina (Al2IO3), CaCO3, Cellulose, Kaolin, MgO, Magnesium Silicate, Silicic acid
(b) Nonpolar: Carbon
It is used for analysis of porphyrins separation.
Technique # 4. Gel Permeation Chromatography:
Gel Permeation Chromatography (GPC) is a liquid chromatographic method known variously as gel filtration and Molecular Exclusion Chromatography (MEC). The gel structure contains pores of varying diameters, up to a maximum size.
The test molecules are washed through a column of the gel and molecules larger than the largest pores in the gel are excluded from the gel structure. Smaller molecules, however, penetrate the gel to a varying extent depending upon their size and this retards their progress through the column.
Elution, therefore, is in order of decreasing size (Fig. 4.1):
There are a variety of products available for Gel Permeation Chromatography (Table 4.2) and they are usually classified according to their exclusion limit, i.e., the relative molecular mass above which all molecules are excluded from the gel structure.
Sephadex, the original medium, is based on dextran (a linear glucose polymer) that is modified to give varying degrees of cross-linking which determines the pore size of the material. It is a strongly hydrophilic polymer and swells considerably in water; before a column is prepared, the gel must be fully hydrated.
Polyacrylamide beads are also available with a wide range of pore sizes and are prepared commercially in a manner similar to that described for polyacrylamide electrophoresis. They are also hydrophilic and swell significantly in aqueous solutions but are chemically more stable than the dextran gels. Agarose gels are particularly useful when gels with a very large pore size are required.
They are also hydrophilic but are usually sold in the swollen form. A major problem is the fact that they soften at temperatures above 30°C.
Mixed gels of polyacrylamide and agarose are available (Ultra-gel, LKB) in which the polyacrylamide provides a three-dimensional structure which supports the interstitial agarose gel. Polystyrene gels are hydrophobic and, as a result, are used primarily with non-aqueous solvents and for organic chemical applications rather than with biological samples.
Technique # 5. Gas Chromatography Principle:
Gas-Liquid Chromatography (GLC) accomplishes a separation by partitioning solutes between a mobile gas phase and a stationary liquid phase held on a solid support. Gas-Solid Chromatography (GSC) employs a solid adsorbent as the stationary phase.
The sequence of a gas chromatographic separation is:
A sample containing the solutes is injected into a heating block where it is immediately vaporized and swept as a plug of vapour by the carrier gas stream into the column inlet. The solutes are adsorbed at the head of the column by the stationary phase and then desorbed by fresh carrier gas.
This sorption- desorption process occurs repeatedly as the sample is moved toward the column outlet by the carrier (mobile phase) gas. Each solute will travel at its own rate through the column. Their bands will separate to a degree that is determined by the individual partition ratios and the extent of band spreading.
The solutes are eluted sequentially in the increasing order of their partition ratios and enter a detector attached to the column exit. If a recorder is used, the signals appear on the chart as a plot of time versus the composition of the carrier gas stream. The time of emergence of a peak is characteristic for each component; the peak ‘area is proportional to the concentration of the component in the mixture.
Although Gas Chromatography is limited to volatile materials — about 15% of all organic compounds — the availability of column temperatures up to 450°C, pyro lytic techniques, and the possibility of converting non-volatile materials into a volatile derivative extend somewhat the applicability of the method.
Basically, a Gas Chromatograph consists of six parts:
(1) A supply of carrier gas in a high pressure cylinder with attendant pressure regulators and flow meters, and a valve to introduce extra make-up gas to some detectors,
(2) A sample injection system,
(3) The separation column,
(4) The detector,
(5) An electrometer and strip-chart recorder (and integrator perhaps), and
(6) Separate thermo-stated compartments for housing the column and the detector so as to regulate their temperature, or to program the column temperature.
The components are shown schematically in Fig. 4.2:
The most exacting problem in Gas Chromatography is presented by the sample injection system. The sample must be introduced as a vapour in the smallest possible volume and in a minimum of time without decomposition or fraction occurring. Generally, two basic type of columns, viz., packed or capillary columns, are used for analytical purpose.
A variety of detector systems is used for analytical purpose, viz., Photoionization (PID), Flame ionization (FID), Electron capture (ECD), Thermal conductivity (TCD) and Flame photometric (FPD).
Among them, thermal conductivity detectors were first and are still widely used; their simplicity is an advantage and they are non-destructive. In contrast — for high-sensitivity analyses of organic compounds — the hydrogen flame ionization detector is used.
Technique # 6. High Performance Liquid Chromatography (HPLC):
This is a specialized version of Column Chromatography, where much more precision analysis of organics will be available. The wide applicability, speed and sensitivity of HPLC have resulted in it becoming the most popular form of chromatography and virtually all types of biological molecules have been analyse or purified using this technique.
There are four distinct modes of HPLC separation methods — Adsorption, Partition, Ion Exchange and Exclusion. All of them vary with the nature of stationary phase.
Some examples of HPLC stationary phases are given in Table 4.2: